Single stranded (ss) DNA-binding proteins are essential to virtually all aspects of DNA metabolism. These proteins, typified by the Escherichia coli ssDNA-binding protein (SSB) in Bacteria (Sancar, A., et al., Proc. Natl. Acad. Sci. USA 78, 4274-4278 (1981), Lohman, T. M. et al, Annu. Rev. Biochem. 63, 527-570 (1994)) and the human replication protein-A (RPA) complex in Eucarya (Fairman, M. P. et al., Embo J. 7, 1211-1218 (1988); Wold, M. S. et al., Proc. Natl. Acad. Sci. USA 85, 2523-2527 (1988); and Wold, M. S., Annu. Rev. Biochem. 66, 61-92 (1997)), are required for in vitro DNA replication and are key components in DNA recombination and repair. Although functionally equivalent, SSB protein and RPA have very different protein structures. Bacterial SSB proteins are encoded by a single gene, although the active form is a homotetramer of SSB where each monomer contributes one ssDNA-binding domain. By contrast, the RPA complex is composed of three distinct subunits.
The large subunit of RPA, RPA70, has several-domains, each associated with a given function (Gomes, X. V., J. Biol. Chem. 270, 4534-4543 (1995), Gomes, X. V. Biochemistry 35, 10558-10568 (1996)). The N-terminal region of RPA70 mediates interactions between RPA and many cellular or viral proteins, whereas the central region contains two functional, homologous, ssDNA-binding sites that are arranged in tandem. The C-terminal region of RPA70 is involved in the assembly of the heterotrimeric complex. The intermediate subunit of RPA, RPA32, which carries a third functional ssDNA-binding site (Bochkareva, E., et al., J. Biol. Chem. 273, 3932-3947 (1998)), is phosphorylated in a cell-cycle dependent manner, although no specific role is attributed to this modification (Henricksen, L. A., Nucleic Acids Res. 24, 3107-3112 (1996)). Finally, the small subunit (RPA14) carries an additional putative ssDNA-binding motif; however, but no direct evidence for DNA binding by this subunit exists.
Interestingly, the four ssDNA-binding motifs of RPA and the motif of SSB protein show a significant degree of homology (Philipova, D. et al., Genes Dev. 10, 2222-2233 (1996)). Moreover, there is striking structural conservation among the ssDNA-binding domains of RPA, and members of both the prokaryotic SSB protein family and the phage-encoded SSB proteins (Bochkarev, A., Nature 385, 176-181 (1997)). These findings suggest that RPA and SSB protein originated from a common ancestral ssDNA-binding protein and then diverged through evolution by a combination of duplications, deletions, and additions.
While ssDNA binding proteins have been known for Bacteria and for Eucarya, the art has not identified a counterpart of a ssDNA binding protein for the third domain of life, the Archaea.